Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 μs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1’s unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

For the closed state, the binding of RAF1 and CDC37 to HSP90 dimer.d.For the closed state, the binding of RAF1 to the CDC37-HSP90 dimer complex.Interaction energies between RAF1 and the rest of the complex (HSP90(A), HSP90(B), and CDC37) and interactions between RAF1 and CDC37 with the HSP90 dimer are also shown.
By comparing the closed and semi-open states using energetic decomposition analysis, we can observe the stabilizing contribution of the CTD (HSP90) towards the formation of the closed-state complex, as well as the contribution of the NTD (HSP90) in stabilizing the HSP90 dimer in both states.Moreover, our comparative analysis reveals a redistribution of vdW contacts, originally present between the MD residues of HSP90 and luminal and peripheral residues of RAF1 in the closed state, to contacts made between the MD residues of HSP90 in the semi-open state.
Through PCA, we were further able to quantify and observe the motions contributing to the variance in our molecular simulation data, noting that the largest uncorrelated motion reveals asymmetric fluctuations in the HSP90 protomers in both the closed and semi-open states.Our simulations support and augment the cryo-EM structural work by providing quantitative analysis of system component interaction energetics through MM-GBSA and pairwise-residue energetic decomposition.The interaction energy values that we provide, and the observations that we draw from the dynamics of the system provide a framework for further mutagenesis experiments and binding assays.Furthermore, our computationally determined RMSF visualizations (Figs.1e, 6f, 6g) can be directly related to the structure factors for each of the solved structures.

6 .
original particles after 2D and 3D Classification De novo initial model Multiple rounds of 3D Classification With de novo initial model 361,383 particles Supplementary Figure 5. Cryo-EM processing workflow.A schematic representation of the data processing workflow in RELION.For details, see the Materials and Methods section of the Image Processing section.Representative cryo-EM maps and masks are depicted at various stages of image processing that yielded both the closed and semi-open states.The number of particles and the global resolution of some of the classes are indicated.The three different reconstructions for the closed state are designated R1, R2, and R3.Characteristics of the map associated with the RAF1-HSP90-CDC37 complex in the closed state.a.The gold-standard Fourier Shell Correlation (FSC) curve for the 3D reconstruction of the closed state.b.Directional FSC histograms and 3DFSC curves.c.Surface representation of the RAF1-HSP90-CDC37 complex, colored to reflect the local resolution calculated by Phenix Local Resolution.d.The angular distribution of the final reconstruction.Each column represents one view, and the size of the column is proportional to the number of particles in that view.

Supplementary Figure 7 .
Characteristics of the map associated with the HSP90 dimeric complex in the semi-open state.a.The gold-standard Fourier Shell Correlation (FSC) curve for the 3D reconstruction of the semi-open state.b.Directional FSC histograms and 3DFSC curves are shown in this panel.c.Surface representation of the dimeric HSP90 complex, colored to reflect the local resolution calculated by Phenix Local Resolution.d.The angular distribution of the final reconstruction.Each column represents one view, and the size of the column is proportional to the number of particles in that view.

Supplementary Figure 8 .
Cryo-EM processing workflow for the semi-open state structure solved using cross-linked sample.A schematic representation of the data processing workflow in RELION.The scale bar represents 50 Å.For details, please see the Materials and Methods section of the Image Processing section.Exemplar 2D class averages and cryo-EM maps are depicted at various stages of image processing that yielded both the closed and semi-open states for the cross-linked sample.The number of particles and the global resolution of various classes are indicated.

Supplementary Figure 9 .Supplementary Figure 10 .
Characteristics of the map associated with the HSP90 dimeric complex in the semi-open state obtained using the cross-linked sample.a.The gold-standard FSC curve for the 3D reconstruction of the semi-open state obtained using the cross-linked sample.b.Directional FSC histograms and 3DFSC curves are shown in this panel.c.Surface representation of the dimeric HSP90 complex obtained using the cross-linked sample, colored to reflect the local resolution calculated by Phenix Local Resolution.d.The angular distribution of the final reconstruction.Each column represents one view, and the size of the column is proportional to the number of particles in that view.Nucleotide-dependent binding of KRAS to RAF1 (RBD-CRD) was studied in the RAF1-HSP90-CDC37 complex.Avi-tagged KRAS (biotinylated) was loaded with either GDP or GMPPNP and immobilized on streptavidin-coated magnetic beads.Subsequently, the closed state complex of RAF1-HSP90-CDC37 was added, and washing and elution steps were conducted to investigate the binding interactions.The results from the pull-down were analyzed using SDS-PAGE, which indicates the presence or absence of each molecular component within the complex.Different lanes on the gel shown in the upper panel represent the specific conditions under which elution and binding interactions were examined.The lower panel shows the uncropped gel, with the boxed part showing the relevant lanes explained in the upper panel.

Supplementary Figure 11 . 12 .Supplementary Figure 13 .
Interactions formed by CDC37 with HSP90 protomers in the closed state.a.This panel shows the interactions formed by CDC37 residues with the two promoters of HSP90 in the closed state.Three dashed rectangles inside this panel depict three different sets of interactions, which are enlarged in panels b, c, and d. b.Interactions formed by the first ten residues of CDC37 with both protomers of HSP90.c. Interactions formed by CDC37 residues 10 through 20 with both protomers of HSP90.d.Interactions formed by CDC37 residues 121-128, forming a beta-strand with the beta-sheet on HSP90 protomer B. Nucleotide-binding site in the N-terminal domain of HSP90 in the closed and semi-open states.a.The overall structure of the closed state complex, showing the electron potential map for the bound nucleotide in the NTD of both protomers of HSP90.b, c.An enlarged view of the ATP (ADP + molybdate) binding pocket in (b) protomer A and (c) protomer B of HSP90, with the electron potential map depicted in gray mesh and the side chain of R391 facing the gamma-phosphate of the ATP.d.The overall structure of the semi-open state complex, showing the electron potential map for the bound nucleotide in the NTD of both protomers of HSP90.e, f.An enlarged view of the ATP (ADP + molybdate) binding pocket in (e) protomer A and (f) protomer B of HSP90, with the electron potential map depicted in gray mesh and the side chain of R391 facing the gamma-phosphate of the ATP.Heatmap showing interactions between residues present in the NTD of HSP90 for both semi-open and closed states.Heatmap showing interactions between key residues for semi-open (a, b) and closed (c, d) states.Residue interactions were identified within 5 Å proximity to the first 26 residues of the NTD in each protomer (engaging in b-strand swap) for both the semi-open and closed states, and combined into a common selection to facilitate direct comparison between the two states.Panels a and c show the heatmaps for the electrostatic interactions, while panels b and d show van der Waals interactions (favorable in blue, unfavorable in red).The most favorable interactions are indicated by black arrows, whereas other notable interactions are shown using gray arrows.

Supplementary Figure 14 .Supplementary Figure 15 .Supplemental Figure 17 .Supplementary Figure 19 .
Heatmap of interactions between the two protomers of the HSP90 dimer that are proximal to the RAF1 luminal insertion and HSP90 src-loop.Heatmap showing HSP90 interactions for key residues in the semi-open (a, b) and closed (c, d) states.Residue interactions were identified within 5 Å of RAF1 luminal residues (418 -426) and proximal RAF1 residues (427, 428), and within 5 Å of the src-loop (residues 340 -350) on each protomer of HSP in the closed and semi-open states, and combined into a common selection to facilitate direct comparison between the two states.Both selections were performed on protonated structures.Panels A and C show the heatmaps for electrostatics interactions, while panels B and D show van der Waals interactions (favorable in blue, unfavorable in red).The green box in all four panels highlights the interaction of the src-loops of the two HSP90 protomers with one another.In panels C and D, the top left and bottom right quadrants formed by horizontal and vertical lines show the interactions formed by CTD residues with residues present in NTD and MD, while the top right quadrant shows the CTD residues from two protomers interacting with one another (panels c, d).The most favorable interactions are indicated by black arrows, whereas other notable interactions are shown using gray arrows.Heat map showing interactions of RAF1 with HSP90 and CDC37 in the closed state complex.Panels a and b show the heatmaps for the electrostatics and van der Waals interactions, respectively (favorable in blue, unfavorable in red).Residue interactions were identified within 5 Å of RAF1 luminal residues (418 -426) and proximal RAF1 residues (427, 428), and within 5 Å of the srcloop (residues 340 -350) on each protomer of HSP in the closed state, and combined into an overall selection.Both selections were performed on protonated structures.The most favorable interactions are indicated by black arrows, whereas other notable interactions are shown using gray arrows.The luminal residues of RAF1 are denoted to the left of the vertical line.Luminal interactions between RAF1 unfolded residues and HSP90.The program LigPlot (J.Chem.Inf.Model., 51, 2778-2786) was used to generate the interactions.RAF1 unfolded luminal residues are shown in purple.Polar interacting HSP90 residues are shown in tan.HSP90 residues that make van der Waals contacts are shown in red semicircles.Labels indicate the residue name and number, as well as what chain they belong to.Chains A, B, and C correspond to HSP90 protomer A, HSP90 protomer B, and RAF1, respectively.The label colors (red, blue, green, and orange) correspond to the src-loop, Helical Hairpin, Helix 16, and Helix 21, respectively, on either HSP90 protomer, and RAF1 labels are colored magenta.HSP90 residues that are not part of the listed secondary structure elements are labeled in black.Structural comparison of the semi-open state HSP90 dimer with TRAP1 NTD-MD dimer.a. Overall structure of the semi-open HSP90 dimer and crystal structure of TRAP1 (PDB 4IVG) NTD-MD dimer shown in the same orientation.b.An enlarged view shows the nucleotide-binding sites in the superposed structures.The semi-open state has ATP, whereas TRAP1 has AMP-PNP modeled in the nucleotide-binding pocket.c.A top-down view displays the src-loop region present in the HSP90 semi-open state structure.This region is disordered in the TRAP1 structure.

Supplementary Figure 22 .
Schematic illustration showing estimated binding energies for four different complex formations.HSP90-A, HSP90-B, RAF1, and CDC37 are colored cyan and blue, magenta, and orange, respectively (same color scheme as in Figure1).Binding energies were calculated by the MM-GBSA method for each replica simulation.a.For the semi-open states (both semi-open and semiopen-cross-linked), the binding of the two HSP90 protomers forming the dimer.The jagged edge indicates the missing C-terminal domain.b.For the closed state, the binding of HSP90 protomers forming the dimer.c.

Energies are in kcal/mol Supplementary Table 1. MM-GBSA analysis to determine the calculated binding free energy between the proteins in closed and semi-open state complexes.
Energies for all three complexes are shown for HSP90(A) with HSP90(B): closed, semi-open, and cross-linked semi-open states.